Cytokines are immunomodulatory proteins, some of which have been used preclinically and clinically not only to fight cancer, but also to interfere with chronic inflammatory conditions and with infectious disease.
The therapeutic potential of recombinant cytokines is often limited by severe side effects even at low concentrations, thus preventing sufficient cytokine concentrations at the target tissues. Recently, monoclonal antibodies have been employed to target and deliver cytokines to sites of disease for increasing their potency and sparing normal tissue from toxic effects. Indeed, a number of antibody-cytokine fusion proteins have already been investigated for application in cancer therapy, often with impressive results. For example, the human antibody L19 specific to the ED-B domain of fibronectin (a marker of angiogenesis) has been used to deliver pro-inflammatory cytokines (such as IL-2, IL-12 or TNF) to solid tumors, sometimes with striking therapeutic benefits [for a review and corresponding references see Neri & Bicknell, Nat. Rev. Cancer (2005) 5:436-446, and also WO 01/62298]. However, many cytokines have a history of clinical failure, both, when used as a single agent or as fusion partners with monoclonal antibodies. For example, recombinant IL-2 (“Proleukin”, Chiron) has been approved for the treatment of patients with renal cell carcinoma but response rates are typically low (generally below 20%) for this indication and even lower for other types of cancer. Other cytokines (such as interleukin-12 or interleukin-10, see below) have failed to demonstrate substantial efficacy in a series of clinical studies which has slowed clinical development programs. These cytokines are not yet approved as biopharmaceuticals. Interferon gamma is another example of a cytokine approved for a very narrow indication (treatment of chronic granulomatous disease, Genentech) which has failed to demonstrate substantial clinical benefits for other indications.
Even when fused to antibodies a striking gain in therapeutic index is unpredictable. For example, the anti-GD2 antibody-IL2 fusion EMD273063 failed to demonstrate substantial therapeutic benefits in a number of clinical trials, last but not least a trial in children with neuroblastoma (Osenga et al., Clin. Cancer Res. March 15; 12(6):1750-9 (2006)).
Interleukin-10 (IL-10) is a homodimeric cytokine produced by activated monocytes and T cells that is deeply involved in the regulation of inflammatory responses and immune reactions. Its main overall function is best described as dampener of immune responses, but IL-10 also possesses stimulatory activities. IL-10 was first described as cytokine synthesis inhibitory factor (CSIF), an activity produced by mouse Th2 cells that inhibited activation of and cytokine production by Th1 cells [Fiorentino et al., J. Exp. Med. 170(6): 2081-95 (1989)]. The gene encoding human IL-10 is located on chromosome 1 [Kim et al., J. Immunol. 148(11): 3618-23 (1992)] and is translated into a protein composed of 160 amino acids with a molecular mass of 18.5 kDa. Human IL-10 is active as a non-disulfide-linked homodimer of 37 kDa [Syto et al., Biochemistry 37(48): 16943-51 (1998)].
IL-10 has been considered an attractive candidate for therapeutic use based on its potent in vitro immunomodulating activities and proven effects in animal models of acute and chronic inflammation, autoimmunity, cancer and infectious disease. Schering-Plough developed recombinant human IL-10 (ilodecakin, Tenovil®) for clinical trials. The protein is produced in E. coli and consists of 161 amino acids, identical with the endogenous human protein except for a methionine residue at the amino-terminus. Phase I and II clinical trials investigating safety, tolerance, pharmacokinetics, pharmacodynamics, immunological and hematological effects of single or multiple doses of IL-10 administered by intravenous or subcutaneous routes have been performed in various settings on healthy volunteers and specific patient populations [Moore et al., Annu Rev. Immunol. 19: 683-765 (2001)]. Clinical development though has been discontinued due to lack of efficacy of the compound. Recently, data has been presented which may explain, at least in part, the dilemma of IL-10 therapy. Tilg et al. found that high doses of IL-10 upregulate the production of IFN-gamma and neopterin, thereby counterbalancing its immunosuppressive properties. The authors concluded that the therapeutic action of systemically administered huIL-10 is limited by proinflammatory effects of the cytokine and suggest that this problem may be circumvented by approaches that result in effective mucosal delivery without causing an increase in systemic IL-10 concentrations [Tilg et al., Gut 50(2): 191-5 (2002)].
Interleukin-15 (IL-15) is a 14 to 15 kDa member of the 4α-helix bundle family of cytokines composed of 114 amino acids. In particular, IL-15 protein is posttranscriptionally regulated by multiple controlling elements that inhibit translation, including 12 upstream AUGs of the 5′ untranslated region (UTR), 2 unusual signal peptides (the short peptide with 21 amino acids stays intracellularly, the long peptide with 48 amino acids is for secretion) and the C-terminus of the mature protein [Bamford et al., J. Immunol., 160(9): 4418-26 (1998)]. There is 97% sequence identity between human and simian IL-15 and 73% between human and mouse. This appears to be sufficient for huIL-15 to render it biologically active on simian and murine cells. IL-15 uses two distinct receptors and signalling pathways: A high affinity IL-15R system consisting of IL-2/15β, γc and IL-15Rα subunits is expressed on T and NK cells. The IL-2/15R β and the γc subunits are shared with IL-2 receptor [Giri et al., EMBO J., 3(12):2822-30 (1994)]. Mast cells respond to IL-15 with a receptor system that does not share elements with the IL-2 receptor but uses a novel 60 to 65 kDa IL-15RX subunit. A variety of tissues such as placenta, skeletal muscles, kidney, fibroblasts, epithelial cells, dendritic cells and monocytes express IL-15.
IL-15 stimulates the production of proinflammatory cytokines (e.g. TNFα, IL-1, IFNγ), the proliferation and Ig synthesis of activated B cells, the activation of TH1, monocytes and lymphokine activated killer cells, the proliferation of mast cells and T cells and inhibits the apoptosis of T and B cells. In addition to the mentioned functional activities IL-15 plays a pivotal role in the development, survival and function of NK cells [Joost J. Oppenheim et al., Cytokine Reference; 213-221, (2002)]. In vivo studies demonstrated that exogenous IL-15 enhances the antitumor activity of tumor reactive CD8+ T cells [Fehniger et al., Cytokine Growth Factor Rev., 13(2):169-83 (2002)].
Abnormal high levels of IL-15 expression have been reported in inflammatory, neoplastic diseases and autoimmune diseases, e.g. rheumatoid arthritis, ulcerative colitis, Crohn's disease and multiple sclerosis [Joost J. Oppenheim et al., Cytokine Reference; 213-221, (2002)].
Because IL-2 and IL-15 use the same receptor subunits they share many features. The major differences are their sites of synthesis and secretion. IL-2 is produced by activated T-cells. In contrast, IL-15 is expressed in a variety of tissues as mentioned above. While IL-2 can promote apoptosis and limited CD8+ memory T-cell survival and proliferation, IL-15 helps maintain memory CD8+ population and can inhibit apoptosis. IL-15, initially thought to mediate similar biological effects as IL-2, has been shown to have unique properties in basic and pre-clinical studies that may be of benefit in the immunotherapy of cancer [Fehniger et al., Cytokine Growth Factor Rev., (2):169-83 (2002)]. Also, the toxicity profile of IL-15 resembles that of IL-2 very closely [Munger et al., Cell Immunol., 5(2):289-93 (1995)], thus suggesting targeted delivery of IL-15 to be superior to systemic delivery in terms of therapeutic index.
Studies to identify the epitopes of IL-15 that are responsible for binding to the IL-15 receptor revealed IL-15 mutants that showed either agonist or antagonist properties which may be useful as therapeutic agents [Bernard et al., J. Biol. Chem., 279(23): 24313-22 (2004)]. The IL-15 mutants IL-15D8S and IL-15Q108S were inactive in a CTLL-2 bioassay, but were able to competitively inhibit the biological activity of unmodified IL-15 [Pettit et al., J. Biol. Chem., 272(4): 2312-8 (1997)].
The melanoma differentiation associated gene-7 (mda-7=IL-24) was first identified in the 1990's as a consequence of its property of being induced during melanoma differentiation. It is a member of the IL-10 family of cytokines. The IL-24 gene cDNA encodes a 206 amino acid protein with 23.8 kDa. In human cells the secreted protein has a significantly higher molecular weight (40 kDa) due to heavy N-glycosylation compared to the intracellular protein (30/23 kDa). The homology of human IL-24 to the rat counterpart (MOB-5) is 68% and to the mouse one (FISP) 69%. There are two functional heterodimeric receptors for IL-24: IL-20R1/IL-20R2 and IL-22R1/IL-20R2 [Wang et al., Genes Immun., 5(5):363-70 (2004)], [Chada et al., Mol. Ther., 10(6):1085-95 (2004)]. Although IL-20R1 and IL-22R1 receptor chains are widely expressed the restricted expression of the common IL-20R2 in certain non-hemopoietic tissues suggests a pleiotropic role of IL-24 outside the hemopoietic system [Wolk et al., J. Immunol., 168(11): 5397-402 (2002)]. IL-24 is expressed by monocytes, T cells, dendritic cells and melanocytes. IL-24 induces the secretion of IFNγ, IL-6, TNFα, IL-1-β and GM-CSF indicating its function as a pro-Th1 cytokine. IL-10 (Th2 cytokine) inhibits the IL-24 activity.
The amount of IL-24 deposit is inversely correlated with melanoma progression. These findings lead to the hypothesis that mda-7 production is lost during melanoma invasion suggesting a role of IL-24 as a tumor suppressor [Chada et al., Mol. Ther., 10(6):1085-95 (2004)].
Expression of IL-24 in tumors may promote antigen presentation by activation or stimulation of immune accessory and effector cells [Chada et al., Mol. Ther., 10(6):1085-95 (2004)].
A large body of data demonstrates that overexpression of the IL-24 gene using either plasmid vectors or a replication defective adenovirus results in growth suppression and induction of apoptosis through activation of intracellular signalling pathways in a broad range of cancer cells. This kind of gene transfer exhibits minimal toxicity on normal cells while inducing potent apoptosis in a variety of cancer cells [Sieger et al., Mol. Ther., 9(3):355-67 (2004)]. A phase I dose escalation clinical trial, where adenoviral constructs expressing the IL-24 were administrated to 22 patients with advanced cancer, resulted in IL-24 expression, induction of apoptosis in all tumors and patients showed increases in CD3+CD8+ T cells after treatment. [Tong et al., Mol. Ther., 11(1):160-72 (2005)]. Different gene transfer studies of IL-24 noted that the tumors were smaller and appeared less vascularized compared to control tumors, which indicates antiangiogenic activity of IL-24 [Saeki et al., Oncogene., 21(29): 4558-66 (2002)]. When using adenovirus mda-7 (Ad-mda7) it is to be noted that there are potential drawbacks for its application in a clinical setting: first of all, ex vivo transduction of human cancer cells obtained from cancer patients with Ad-Mda7 followed by reintroduction into cancer patients is not practical; secondly, intratumoral administration of Ad-mda7 to generate a potent antitumor immune response is applicable only to localized tumors and not for disseminated tumors. Thus, alternative approaches need to be developed [Miyahara et al., Cancer Gene Ther. 2006].
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 141 amino acid (mouse)/144 amino acid (human) protein containing a 17 amino acid secretion sequence. The apparent molecular weight of the mature glycosylated protein is 14-33 kDa, which is very resistant to denaturing and proteolytic conditions. The in vivo activities of GM-CSF are mediated by binding to high-affinity receptors comprising a GM-CSF-specific α chain and, for humans, a signal transducing β subunit that is shared with the IL-3 and the IL-5 receptors [Joost J. Oppenheim et al., Cytokine Reference, 899-908, 2002].
GM-CSF is a major regulator of granulocyte and macrophage lineage. It stimulates the survival, proliferation and differentiation of hematopoietic colony-forming cells of the neutrophil, macrophage and eosinophil lineages. In addition, it maintains the survival of hematopoietic colony-forming cells of the megakaryocytic and erythroid cell lineages [Joost J. Oppenheim et al., Cytokine Reference, 899-908, 2002]. It is also a potent immunostimulator with pleiotropic effects, including the augmentation of Ag presentation in a variety of cells, increased expression of MHC class II on monocytes and amplification of T cell proliferation [Fischer et al., J. Immunol., 141(11):3882-8 (1988), Smith et al., J. Immunol., 144(10):3829-34 (1990), Morrissey et al., J. Immunol., 139(4):1113-9 (1987)].
In pathology overexpression of GM-CSF may lead to inflammatory reactions (e.g. rheumatoid arthritis), toxic shock, blindness and autoimmunity while subphysiological levels may be involved in some cases of alveolar proteinosis. Alveolar proteinosis is a fatal lung disease where surfactant proteins accumulate in the lung due to a defect in macrophage-mediated clearance [Joost J. Oppenheim et al., Cytokine Reference; 899-908, 2002].
In animal models vaccination of mice bearing B16 melanoma with additional irradiated tumor cells expressing murine granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated a potent, long-lasting and specific anti-tumor immunity by increasing the immunogenicity of the tumors [Dranoff et al., Proc. Natl. Acad. Sci. USA., 90(8):3539-43 (1993)]. Additionally, GM-CSF is widely used in oncology to reduce chemotherapy-related neutropenia, a reduction of neutrophils caused by chemotherapeutic drugs [Danova et al., Haematologica., 82(5):622-9 (1997)], Nose et al., J. Clin. Oncol., 13(4):1023-35 (1995)]. There is a threshold above which a GM-CSF based vaccine not only loses its efficacy but more importantly results in substantial immunosuppression in vivo. The dual effects of GM-CSF are mediated by the systemic and not the local concentration of this cytokine [Serafini et al., Cancer Res., 64(17):6337-43 (2004)]. Serious adverse events are seen at doses of 16 μg/kg per day for humans [Joost J. Oppenheim et al., Cytokine Reference; 899-908 (2002)].
Fibronectins are high molecular weight adhesive glycoproteins present in soluble form in plasma and other body fluids and in insoluble form in the extracellular matrix. EDB is a 91-amino-acid type III homology domain that is inserted into the fibronectin molecule by a mechanism of alternative splicing at the level of the primary transcript whenever tissue remodelling takes place [Zardi et al., Embo J. 6(8): 2337-42 (1987)].
EDB is essentially undetectable in healthy adult tissues. Its expression is strongly associated with the remodelling of the extracellular matrix and angiogenesis. The domain is abundant in many aggressive tumors and depending on the tumor type displays either predominantly vascular or diffuse stromal patterns of expression [Carnemolla et al., J. Cell Biol. 108(3): 1139-48 (1989)]. Despite its very restricted expression in normal tissues and its strong expression in many solid tumors the function of EDB does not seem to be indispensable because mice lacking the EDB exon develop normally, are fertile and heal bone fractions. Furthermore, double knock-out mice lacking the EDB exon and p53 did not show any difference in the duration of survival compared to animals expressing EDB [Fukuda et al., Cancer Res 62(19): 5603-10 (2002)].
Because the EDB sequence is identical in mouse, rat, rabbit, dog, monkey and man it has not yet been possible to raise antibodies against this domain by hybridoma technology due to natural tolerance. A few years ago high affinity scFv antibody fragments (L19) against EDB were isolated by phage display technology [Carnemolla et al., Int. J. Cancer 68(3): 397-405 (1996); Neri et al., Nat. Biotechnol. 15(12): 1271-5. (1997); Pini et al., J. Biol. Chem. 273(34): 21769-76 (1998)]. L19 is able to stain tumor blood vessels in a wide range of experimental tumor models and on sections of human tumors and other angiogenic disorders [Carnemolla et al., J. Cell Biol. 108(3): 1139-48 (1989); Kaczmarek et al., Int. J. Cancer 59(1): 11-6 (1994); Berndt et al., Histochem. Cell Biol. 109(3): 249-55 (1998)]. Castellani et al. have shown that L19 stains tumor blood vessels in grade III-IV astrocytomas but less than 10% of the vessels in grade I-II astrocytomas, suggesting that the expression of EDB in these lesions could be used for grading of the tumors [Castellani et al., Am. J. Pathol. 161(5): 1695-700 (2002)].
Due to the conservation of the antigen the targeting performance of L19 could be investigated in immunocompetent syngeneic animal models. Biodistribution studies with different radiolabelled antibody formats (scFv, small immuno protein/SIP and IgG) showed a preferential accumulation of up to 20% injected dose per gram of tissue (% ID/g) of L19 at the tumor site [Borsi et al., Blood 102(13): 4384-92 (2003)]. First immunoscintigraphy studies in human cancer patients with L19-diabody labelled with 123I confirmed that the antibody also localizes to human solid tumors and metastases [Santimaria et al., Clin. Cancer Res. 9(2): 571-9 (2003)].
The EDB domain of fibronectin is a good-quality marker of angiogenesis, which is overexpressed in a variety of solid tumors (e.g., renal cell carcinoma, colorectal carcinoma, hepatocellular carcinoma, high-grade astrocytomas, head and neck tumors, bladder cancer, etc.) but is virtually undetectable in normal adult tissues (exception made for the endometrium in the proliferative phase and some vessels in the ovaries). However, EDB is only weakly expressed in most forms of breast cancer, prostate cancer and some types of lung cancer, thus stimulating the search for novel vascular tumor antigens, which could be used for the antibody-mediated targeted delivery of therapeutic cytokines to these neoplasias.
In addition to EDB the extracellular domains of oncofetal tenascin have been established as an interesting target in therapy. Splice isoforms of tenascin-C are considered targets for antibody-based therapeutic strategies, particularly for those tumor classes in which low levels of EDB can be detected. Tenascin-C is a glycoprotein of the extracellular matrix. It comprises several fibronectin type 3 homology repeats that can be either included or omitted in the primary transcript by alternative splicing, leading to small and to large isoforms that have distinct biological functions. While the small isoform is expressed in several tissues the large isoform of tenascin-C exhibits a more restricted expression pattern. It is virtually undetectable in healthy adult tissues but is expressed during embryogenesis and is again expressed in adult tissues undergoing tissue remodelling including neoplasia. Its expression is localized around vascular structures in the tumor stroma of a variety of different tumors including breast carcinoma, oral squamous cell carcinoma, lung cancer, prostatic adenocarcinoma, colorectal cancer or astrocytoma and other brains tumors. Traditionally, the scientific community referred to the large isoform of tenascin-C for tenascin molecules, which would putatively comprise all alternatively spliced domains, and to the small isoform of tenascin-C whenever these domains were absent. Carnemolla and colleagues reported that the alternatively spliced domain C of tenascin-C exhibited a more restricted pattern of expression when compared to other alternatively spliced domains. It remained unclear at that time whether other alternatively spliced domains of tenascin-C also exhibited restricted incorporation into the tenascin molecule, and whether it would be more appropriate to evaluate the individual spliced domains separately as targets for antibody-based therapeutic strategies. Radiolabelled antibodies specific for domains A1 and D of tenascin-C were successfully employed in the clinic for the treatment of glioma and lymphoma. Furthermore, efficient tumor targeting by anti-tenascin antibodies has been demonstrated clinically using an avidin/biotin-based pre-targeting approach or, more recently, with monoclonal antibodies specific for the small isoform of tenascin-C. However, all these antibodies are of murine origin and, therefore, are most probably not suitable for repetitive administration to human patients and the development of biopharmaceuticals. For these reasons human antibodies specific to domains A1, C and D of tenascin-C were generated using antibody phage technology [PCT/EP2005/011624 of Philogen S.p.A].
As demonstrated above, there is still a high uncertainty involved in the field regarding the therapeutic utility of cytokines in general, in particular the therapeutic utility of cytokines for treating tumours and/or inflammatory diseases. Although the prior art sporadically indicates that some specific antibody-cytokine fusion proteins might allow for target-directed therapeutic treatment, there is still no reasonable expectation of success because the results are not predictable. The skilled person is left guessing with respect to the nature of a therapeutically useful cytokine and the effect that its combination with an antibody or derivative thereof would have. Therefore, the skilled person requires inventive skill to select the right combination of the many known cytokines and the many known targeting antibodies because the outcome cannot be predicted.
It is the object of the present invention to provide novel therapeutic substances for the treatment of cancer and/or inflammatory diseases, in particular for treating psoriasis, atherosclerosis and arthritis, that allow for the targeted delivery of the therapeutic substance to the sites of disease, which in turn allows for concentrating the medicament and reducing the toxic load for the remaining healthy tissues.